Project Summary: Ras proteins play a central role in many aspects of biology. These monomeric GTPases cycle between an inactivate GDP-bound state and an active GTP-bound state. Mutational activation of Ras occurs in approximately 30% of human tumors with some cancers such as pancreatic cancer having Ras mutations in >90% of tumors. These cancer associated mutations trap Ras in the GTP-bound state, leading to chronic activation of Ras-regulated signaling pathways and oncogenic transformation. Thus, RasGTP has been considered the only biologically important form of the molecule. However, we have discovered a novel role for nucleotide-free Ras (nfRas) in the negative regulation of cell signaling. This form of Ras, formed as an intermediate in the transition from RasGDP to RasGTP, negatively regulates phosphatidylinositol 3-kinase, class 2beta (PI3KC2?). These finding have profound implications for the understanding of Ras-mediated signaling and transformation. We propose that nfRas binds targets, e.g., PI3KC2?, leading to the mutual inhibition of both Ras and the associated target. Our model predicts that oncogenic activation of Ras leads to loss of repression of these targets resulting in their activation without binding RasGTP. Thus, our findings point to a new class of molecules important for Ras-driven tumorigenesis yet do not bind activated Ras. To better understand the role of nfRas in cell signaling, we have generated high affinity, high specificity monobody reagents that selectively bind different states of Ras. Using these reagents we demonstrate that nfRas represents a significant pool of total cellular Ras. In addition, one of our Ras monobodies which binds to all three forms of Ras (GTP, GDP, and nucleotide free) acts as a highly potent inhibitor of oncogenic Ras- mediated signaling. Using these reagents we will characterize the regulation of nfRas in vivo and determine the role of nfRas in regulating specific targets. Finally, we will utilize these novel monobody reagents to perturb Ras function in vivo using a novel chemical-genetic approach to interfere with pancreatic cancer development and progression. Given the prevalence of Ras mutations in human cancers, it is critical to understand the mechanisms through which Ras contributes to tumorigenesis. Our work represents an entirely new concept in Ras biology that defines a new class of targets that participate in Ras-mediated signaling and transformation. These studies will provide new insights into the mechanisms of Ras-mediated tumorigenesis and are therefore of high translational significance. This work will be beneficial to Veterans as well as the general population, both of which suffer from cancer. However, the incidence for certain cancers, such as lung and pancreatic cancer the latter of which has a high incidence of Ras mutations (<90%), has been reported to be up to 5-7x higher in Veteran populations making these studies particularly relevant to Veterans. Additionally, the goals of our proposal are consistent with the recently established National Cancer Moonshots Program to accelerate research efforts to improve cancer treatment. Finally, given the ubiquity of Ras family members in eukaryotes and their roles in many aspects of biology these results will be of broad scientific interest and importance to many areas of biomedical research.